The invention concerns a method for sample picking on cryosubstrates, particularly a method for transferring samples in a cryopreserved or thawed condition from a cryosubstrate to a target substrate. The invention also concerns a device for implementing a method of this type and a cryosubstrate which is functionally textured for sample taking.
The operation of cryobanks for preserving biological cell material is generally known in cell biology, molecular biology, and genetic engineering. In a cryobank, the cell material is kept available for decades, with, for example, suspended cells being frozen in small-volume containers (volumes range from 0.1 ml to a few ml) filled with a cryoliquid. In order to ensure the viability of the cell material after thawing, numerous procedures have been developed which, for example, relate to the timing of the thawing, media additives, container shapes, and similar things. With conventional cryobanks, survival rates ranging from a few percent up to 90% are achieved during thawing. Although this is already a relatively good result and cryobanks have found worldwide distribution, the following disadvantages are connected with the cryopreservation procedures disseminated until now.
The position of individual cell material samples in the volume of the cryoliquid is unknown during both the freezing and the thawing procedures. The material samples are not accessible in the preserved, deep frozen state. However, there is interest in, for example, removing single cells from cryopreserved material, measuring, or changing them. However, in order to be able to remove cells, the entire sample must be thawed. This requires costly recultivation of the cell material to compensate for the thawing losses. Over the course of time, the cryopreserved material thus no longer contains only the originally preserved cells, but a mixture of daughter cells of greatly varying generations, which restricts the specificity and reproducibility of cell investigations. In order to subject all material samples in a cryocontainer to the same cooling progression, extremely slow freezing procedures must be provided, since the cooling proceeds from the container walls and all samples in the cryovolume are to experience approximately the same temperature progression over time. Finally, the suspension medium (cryoliquid) prevents or makes more difficult measurement and processing of single cells at low temperatures.
There is an interest in new cryopreservation technologies for overcoming the disadvantages mentioned and for opening new fields for cell preservation, particularly since researches in biotechnology, genetic engineering, and medicine are increasingly directed toward single cells, such as in hybridoma cell production in connection with tumor treatment, stem cell culture, and embryogenesis. The development of new cryopreservation technologies is based on the following knowledge and considerations.
From a physical and physiological viewpoint, a cell frozen at xe2x88x92196xc2x0 C. is in a solid state. The metabolic processes have come to a complete standstill down to the molecular level. Cell changes only arise through slow restructuring (e.g. through the growth of ice crystals at temperatures above xe2x88x9280xc2x0 C.) and through damage due to cosmic radiation. The latter, however, has a rate of approximately 90% damage after 30,000 years, which is non-critical for practical applications. In the deep frozen state, cells should therefore able to be measured, treated, changed, sorted and otherwise manipulated in a mechanically robust way without time pressure and with the highest precision. However, this assumes the ability to individually handle the cells in the cryomedium and the availability of tools for cell manipulation.
The physical and chemical procedures during the freezing or thawing of biological materials are, for example, described in the publication of F. Franks xe2x80x9cBiophysics and biochemistry of low temperature and freezingxe2x80x9din xe2x80x9cEffects of Low Temperatures on Biological Membranesxe2x80x9d(Editor G. J. Morris et al., Academic Press, London, 1981) or P. Mazur in xe2x80x9cAnn. N.Y. Acad. Sci.xe2x80x9d, vol. 541, 1988, p. 514 et seq. The prevention of the formation of intracellular or extracellular ice crystals and excessive dehydration of the cells is decisive for freeze preservation over long periods of time and thawing with the greatest possible survival rate. In this case, the following characteristics are to be taken into consideration from a physical viewpoint during freezing and thawing. Producing so-called vitrified water, in which any type of ice crystal formation is suppressed, through extremely high freezing speeds is known. However, this cannot be used for careful and positionally defined freezing of cell material, since the size of the biological cells of interest and heat conduction restricts the freezing speeds to values below a few ten thousands of degrees per second. Therefore, at the microscopic scale and under physiological conditions, segregation, i.e. formation of eutectic phases, which also include domains of pure ice, can be observed. To minimize the segregation, cell-specific freezing programs have provided the best results, particularly at the beginning of cooling (down to xe2x88x9230xc2x0 C.), (see also S. P. Leibo et al. in xe2x80x9cCryobiol.xe2x80x9d, vol. 8, 1971, p. 447 et seq). In this temperature interval, cooling rates of a few degrees per minute have been shown to be more favorable than rapid temperature jumps. It is inferred from this that the cooling and thawing procedures should be performed with a biologically specific temperature profile over time.
As soon as temperatures at which ice formation begins have been reached, however, higher cooling rates are appropriate, since in this way the migratory growth of larger ice domains at the cost of smaller ones can be prevented. At temperatures below the range of xe2x88x9280xc2x0 C., no further ice crystal growth occurs, so that cell storage over long periods of time is possible. The storage of the container with cell material which is suspended in a cryoliquid is typically performed in liquid nitrogen (at xe2x88x92196xc2x0 C.). Since the sample container is closed, there is no direct contact with the liquid coolant phase. Comparable temperature sequences are used for thawing the cell material.
Cooling procedures are also known from preparation for electron microscope recordings (see D. G. Robinson et al. in xe2x80x9cPrxc3xa4parationsmethodik in der Elektronenmikroskopiexe2x80x9d, Springer-Verlag, Berlin, 1985). In contrast to cryopreservation, which has the goal of maintaining the vitality of the cells, in electron microscopy, the least possible change in the molecular position of the cell components plays the decisive role. Therefore, during this preparation, particularly rapid freezing technologies are realized, which include, for example, shooting the sample into liquid or undercooled gases or spraying drops into an undercooled atmosphere and liquids. In this case, cooling rates of more than 10,000 degrees per second are achieved, which, however, due to the cell volume, the finite thermal conductivity, and the wettability of the material, represent a limiting value.
A general problem in cryopreservation is that not all types of cells can be cryopreserved in the same way. In particular, larger objects (cell groups or the like) or cells containing large numbers of vacuoles, which particularly occur in plant sample material, can be revitalized only with difficulty or not all. The development of new microinjection and cell handling technologies, as well as new cryoprotectives, is directed toward these problems. A technology which is different from the preservation in containers described above is based on the freezing and/or thawing of the cell material to be preserved in adhered form on cooled surfaces (see, for example, T. Ohno et al in xe2x80x9cCryotechnol.xe2x80x9d, vol. 5, 1991, p. 273 et seq).
Cryopreservation on cooled surfaces is more difficult to handle than the suspension principle, but has been shown to have advantages in the investigation of the processes occurring during cryopreservation and in achieving higher survival rates during thawing. Cryopreservation on substrate surfaces allows the boundary conditions of the respective procedure, such as surface temperature, thermal conduction, cell or droplet size, etc., to be adjusted and detected more exactly and more variably than in the suspension of a cryomedium. This is particularly used in cryomicroscopy, with biological cells which are enclosed in the solvent drops being misted or sprayed onto frozen surfaces (see H. Plattner et al in xe2x80x9cFreeze-etching, Techniques and Applicationxe2x80x9d, editor E. L. Benedetti et al, xe2x80x9cSoc. Franc. Microsc. Electroniquexe2x80x9d, Paris 1973, p. 81 et seq, and PCT/US94/01156). A disadvantage of the initially developed cryopreservation on substrate surfaces is that the position and arrangement of the cells cannot be controlled during misting or spraying and multiple drops and cell layers can even be deposited on top of one another.
An improvement of cryopreservation on substrate surfaces is described in EP 804 073. Biological cells surrounded by an enveloping solution are placed using a microdroplet jetting device in a predetermined way on substrates the temperature of which can be adjusted. The microdroplet jetting device, which can be driven like an inkjet printer, allows a highly precise and reproducible positioning of individual material samples on the cryosubstrate. Texturing the cryosubstrate with recesses applied in a matrix in order to allow specific procedures during cryopreservation and/or during thawing of the substrate is also known from EP 804 073. The recesses are thus particularly adapted for directed deposition of the cells. To produce test chips, with which the interaction of greatly differing cells in the thawed state is to be investigated, various cell types are deposited in or between the recesses. Furthermore, providing electrodes for implementing high frequency electric fields at the recesses, under whose effect an investigation of the cells in the thawed state can be performed, is known from EP 804 073.
Cryopreservation on cooled surfaces has had the disadvantage until now that, after the application onto the cryosubstrate, a sample-specific handling of single cells was only possible in the deep frozen or thawed state on the cryosubstrate. If processing in the thawed state was intended, the entire substrate had to be heated. However, for improvement of the investigation techniques and increased utilization of cryopreserved sample stocks, it is important to make the individual material samples accessible to specific handling.
The object of the invention is to provide an improved method for sample picking on cryosubstrates which particularly allows selective taking of preselected samples or sample groups from a cryosubstrate. The object of the invention is also to provide devices for implementing a method of this type.
These objects are achieved by a method and/or devices with features according to the patent claims, and/or. Advantageous embodiments and applications of the invention arise from the dependent claims.
According to the invention, predetermined, selective sample picking occurs on a cryosubstrate with multiple samples which are located on predetermined sample positions through positionally specific mechanical or thermal separation of the samples from the cryosubstrate and transfer of the released samples to a target substrate. Sample picking is hereby generally understood to mean any type of picking or taking of samples, if necessary with certain parts of the substrate.
Any device which is suitable as a carrier for samples frozen onto cooled surfaces is referred to in this case as a cryosubstrate (or: carrier substrate, substrate). It serves for sample preservation or storage. The cryosubstrate includes a carrier material for arranging the samples in linear or planar shapes with a functional surface texture described in detail below. According to a preferred embodiment, the carrier material consists of an inert material, such as plastic, ceramic, metal, or semiconductor material, which can be structured with a mechanical or chemical processing means known per se. The cryosubstrate preferably forms a rigid, planar, flat or curved body which is bonded in a way known per se with a temperature adjusting device. Alternatively, the cryosubstrate can, however, also be made of a flexible, film-like carrier material, for example plastic.
The carrier material is preferably implemented integrally with the surface texture (or: structure), but can also include a combination of the materials described in specific embodiments. This combination can, for example, be an electrically insulating base material with specific surface coatings made of, for example, metal. For the realization of the present invention, a functional surface texture (or: structure) is generally understood to mean any type of geometrical or material change of the cryosubstrate through which localized deposition regions are created, corresponding to the sample positions on the cryosubstrate, from which the respective sample or samples can be selectively removed without the entire cryosubstrate having to be heated. The sample picking according to the invention therefore preferably occurs on cryosubstrates in the deep frozen operating state.
The method according to the invention can be implemented with any type of sample desired which can be applied onto a cryosubstrate while deep frozen (e.g. at the temperature of liquid nitrogen). The samples preferably consist of biological material, such as biological cells or cell groups or cell components, if necessary each with an enveloping medium. The invention can, however, also be used with synthetic materials, such as vesicles, or with combinations of biological and synthetic materials.
The sample picking and transfer according to the invention occurs toward a target substrate, which refers to, in general, any type of device for further handling or manipulation of the sample. For example, storage, mechanical or chemical processing, or investigation of the sample occurs on the target substrate. The target substrate can thus also be a cryosubstrate of a further preservation system.
A positionally selective mechanical separation of samples from the cryosubstrate includes separation of predetermined deposition elements from the substrate with the respective samples or sample groups. The separation occurs with a suitable tool, preferably while maintaining the cryopreserved state of the samples. However, a mechanical separation in the thawed sample state can also be provided. A thermal separation occurs according to a first embodiment of the invention through a positionally specific increase of the substrate temperature in such a way that the appropriate sample is thawed and removed with a suitable tool (e.g. micropipette, picking needle), or that positionally specific deposition elements with preserved samples are thermally separated from the substrate. For thermal separation, electrical resistance heating or radiation heating (laser, microwaves, or the like) is used on the desired sample position. In an alternative form of thermal sample separation, freezing of the desired samples onto a textured tool, which produces stronger adhesion of the frozen samples than the adhesion to a sample carrier, is provided.
A subject of the invention is also a sample picking or sample handling system for picking and/or transferring samples from a cryosubstrate onto the target substrate, with a system of this type particularly including a functionally textured cryosubstrate, a separation device, and a control device. The separation device serves as a separation device and/or as the picker for the separated or released sample.
According to a particularly important aspect of the invention, a cryosubstrate is provided with a functionally textured surface which includes multiple deposition elements (e.g. deposition plates, deposition films), which are each implemented for accommodating one material sample and for selective mechanical or thermal separation of the sample, if necessary with a part of the deposition element, from the cryosubstrate. The dimensioning of the deposition elements is selected depending on the application. A deposition element can have characteristic dimensions of a magnitude from 1 cm2 to 1 mm2 or even less. The separation of entire sample groups from the cryosubstrate can also be provided.
The invention has the advantage that for the first time the restrictions of cryopreservation on temperature stabilized substrate surfaces are overcome and selective processing of individual samples is made possible. In this way, the effectiveness of single cell cryopreservation is significantly increased. The design of a cryosubstrate according to the invention is based on well controllable texturing methods that are known per se. The cryosubstrate can be produced as a disposable product from economical material. A further advantage concerns the ability to automate the overall system. Through the combination of sample picking with an image processing system, sample transfer from a cryosubstrate to one or more target substrates can be performed independently of the operator and automatically.